Breast Cancer Research and Treatment 50: 63-71. 1998.
`© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
`
`Report
`
`The effects of aromatase inhibitors and antiestrogens in the nude mouse
`model
`
`Qing Lu, Wei Yue, Jiping Wang, Yang Liu, Brian Long, and Angela Brodie
`Department of Pharmacology & Experimental Therapeutics, and Greenbaum Cancer Center, School of Med(cid:173)
`icine, University of Maryland. Baltimore, Maryland, USA
`
`Key words: breast cancer, nude mice, antiestrogens, aromatase inhibitors
`
`Summary
`
`The effects of antiestrogens, tamoxifen and ICI 182,780, and aromatase inhibitors, arimidex (anastrozole
`ZD1033) and letrozole (CGS 20,267), on the growth of tumors were studied in nude mice. In this model,
`estrogen dependent MCF-7 human breast cancer cells stably transfected with the aromatase gene were in(cid:173)
`oculated in four sites per mouse. Sufficient estrogen is produced from aromatization of androstenedione
`supplement (0.1 mg/mouse/day) by the cells to stimulate their proliferation, tumor formation, and maintain
`the uterus similar to that of the intact mouse. Once the tumors reached a measurable size, the mice were
`injected with antiestrogen or inhibitor for 35-56 days. Tumor volumes were measured weekly. At autopsy, the
`tumors were removed, cleaned, and weighed. Statistical data was determined from tumor weights. Both anti(cid:173)
`estrogens were effective in reducing tumor growth in these mice. Tamoxifen appears to be more effective than
`ICI 182,780, although the former stimulated the uterine weight whereas the pure antiestrogen did not. How(cid:173)
`ever, both aromatase inhibitors were more effective than the antiestrogens. Tumor regression was observed
`with letrozole. Thus, after-treatment tumor weights were less than those of a group of mice at the start of
`treatment. The aromatase inhibitors also reduced the weight of the uterus, suggesting that these compounds,
`as well as the pure antiestrogen, may not cause endometrial proliferation.unlike t8moxifon. These :irnm::it::ise
`inhibitors may not only benefit patients who have relapsed from tamoxifen, but may be more effective in
`patients as first line agents for suppressing the effects of estrogen.
`
`Introduction
`
`Breast cancer is the most common malignant neo(cid:173)
`plasm in U.S. women. Although causes of breast
`cancer are not well understood, the contribution of
`estrogens to the development of the normal breast
`and the growth of breast cancer has been widely
`documented [1, 2]. Approximately one-third of hu(cid:173)
`man breast cancers are estrogen dependent. The
`
`dependence of breast tumors on estroge.ns increas(cid:173)
`es with age. Thus, tumors of postmenopausal pa(cid:173)
`tients are more likely to be dependent on estrogens
`for their progression than those of younger women.
`Estrogen receptor positive tumors are generally
`more differentiated. slower growing. and indicate a
`more positive prognosis for the patient. Estrogens
`are synthesized by aromatization of androgen sub(cid:173)
`strates via a series of reactions catalyzed by cyto-
`
`AJJre,sfot offprints and correspondence: A. Drodie. Department of Pharmacology&. Experimental Therapeutics. 655, W. Baltimore St.,
`School of Medicine, University of Maryland, Baltimore, MD 21201, USA: Tel: 410-706-3137: f/1x: 410-706-0032;
`E-mail: abrodie@umarylancl.edu
`
`MYLAN PHARMS. INC. EXHIBIT 1014 PAGE 1
`
`

`
`64
`
`Qing Lu et al.
`
`chrome P450 aromatase (P-450arom). Following
`menopause, when the ovary is no longer the main
`source of estrogen, production is increased in pe(cid:173)
`ripheral tissue, such as adipose and muscle which
`make up most of the body mass. Thus, systemic ap(cid:173)
`proaches to treatment rather than surgical removal
`of adrenals or the pituitary, are proving more effec(cid:173)
`tive, well tolerated, and associated with less mor(cid:173)
`bidity and mortality. For patients who have estro(cid:173)
`gen and/or progesterone receptor positive tumors.
`endocrine therapies are more successful than che(cid:173)
`motherapy [3].The effect of estrogens on the
`growth of breast cancers can be blocked by two ma
`nipulations: inhibition of estrogen action by anties(cid:173)
`trogens, which interact with estrogen receptors [ 4],
`and blockade of estrogen synthesis by inhibitors of
`aromatase [5].
`Tamoxifen (ICI 46,474 ). a nonsteroidal antiestro(cid:173)
`gen, was developed in 1967 and entered clinical
`trials for advanced breast cancer in 1971 [6]. Its use
`as an adjuvant to surgery and as the first line ther(cid:173)
`apy for advanced disease in postmenopausal pa(cid:173)
`Litmts is nuw e~LalJlished [3]. Tamoxifen competes
`with estrogen for binding to the estrogen receptor,
`and blocks the action of estrogen [4]. Adjuvant ta(cid:173)
`moxifen therapy for early breast cancer (both node
`positive and node negative) produces reduction in
`annual rates of recurrence and death (25 % for re(cid:173)
`currence and 17% for mortality). Tamoxifen also re(cid:173)
`duced the risk of development of contralateral
`breast cancer by 39% [7]. However, tamoxifen is a
`weak or partial agonist and for this reason it may
`not be the optimal agent for inhibiting hormone de(cid:173)
`pendent tumor growth. It has also been reported
`th::it tamoxifen treatment increases the risk of de(cid:173)
`veloping endometrial carcinoma, and the incidence
`is correlated with the duration of treatment [8]. This
`effect is thought to be due to the partial agomstic
`action of tamoxifen which in rodents produces a
`uterotrophic response [9].
`In the late 1980's, steroidal antiestrogens were
`developed (10-13) 'vvhich are more potent than ta(cid:173)
`moxifen as antiestrogens and are without agonistic
`effects. In patients with breast cancer, faslodex ( ICI
`182,780:
`7 a-(9-( 4,4,5,5,5-pentafluoropentylsulfi(cid:173)
`nyl)nonyl]estra-1,3,5(10)-triene-3.17~-diol) reduces
`ER content in the tumors and the concentration of
`
`two estrogen regulated proteins, progesterone re(cid:173)
`ceptor and pS2 [13]. Clinical trials with ICI 182,780
`in breast cancer patients are in progress [14].
`In the early 1970's, our laboratory reported the
`first of a series of compounds which selectively in(cid:173)
`hibit estrogen synthesis [15J. A number of steroidal
`agents were found to be potent aromatase inhib(cid:173)
`itors and subsequently 4-hydroxyandrostenedione
`was shown to be effective in treating breast cancer
`[16-19]. Recently, several non-steroidal selective in(cid:173)
`hibitors have been developed which are imidazole
`and triazole derivatives based on antifungal agents
`that inhibit P-450 enzymes. Two triazole com(cid:173)
`pounds have recently been approved as second line
`agents in the treatment of advanced breast cancer in
`postmenopausal patients. They are arimidex
`
`(ZD 1033:2,21[ 5-(lH-1,2,4-triazol-1-yl methyl )-1,3-
`-phcnylcnc ]bis(2-mcthylpropiononitrilc) and
`le(cid:173)
`trozole (CGS 20267: 4-[1-(cyanophenyl)-1-(1,2,4-
`triazolyl)methyl]benzonitrile). Both are very po(cid:173)
`tent and selective aromatase inhibitors and are well
`tolerated by patients. Letrozole ( CGS 20267) is ap(cid:173)
`proximately 100 times more potent than fadrozolc
`(CGS 16949A an imidazole analogue) in reducing
`serum estradiol levels, which may be due to the sig(cid:173)
`nificantly longer half-life of the former in patients
`[20]. Doses of 1to10 mg arimidex have been report(cid:173)
`ed to reduce plasma eslradiul levels tu the limit of
`the detection assay in postmenopausal breast can(cid:173)
`cer patients [21]. Recent clinical studies that com(cid:173)
`pared these inhibitors to other second line agents,
`found that letrozole caused more complete and par(cid:173)
`tial tumor responses than aminoglmethimide [22]
`and that arimidex increased survival of patients to a
`significantly greater extent than megace (23]. How(cid:173)
`ever, the optimal use of these well-tolerated agents
`remains to be determined.
`Because of its established efficacy, most patients
`receive tamoxifen as a first line agent. Thus, direct
`comp8risnn with new agents is ;:i difficult and slow
`process. We, therefore, established a model for
`postmenopausal, hormone-dependent, breast can(cid:173)
`cer in nude mice in which antiestrogens and aroma(cid:173)
`tase inhibitors could be compared [24 ]. In this mod(cid:173)
`el. estrogen receptor positive human breast cancer
`cells (MCF-7) transfected with the aromatase gene
`are inoculated into ovariectomized nude mice.
`
`MYLAN PHARMS. INC. EXHIBIT 1014 PAGE 2
`
`

`
`Aromatase inhibitors and antiestrogens in nude mouse model
`
`65
`
`A
`Ql
`E
`::::i
`r-- control
`0
`>
`0 200
`E
`::::i
`re<:::::=: .c ,./
`I-
`-0
`~ 150
`
`/
`/
`.---: ..
`_.( /
`
`ICI 0.07 mg/w
`_,.,A<-~--A
`/" ICI 0.7 mg/w
`.
`
`------.,,._
`
`. ...--
`
`300 r-
`
`250.
`
`100
`
`50
`
`'----
`0
`
`7
`
`400 1-
`
`350
`
`Ql
`Ol
`c
`ro
`..c u
`
`-<f!_
`
`B
`
`A
`
`350
`
`200
`
`Ql
`E 300
`::::i
`~
`0 250
`E
`::::i
`I-
`]§
`~ 150
`0
`Ql
`Ol 100
`c
`ro
`..c u
`
`~ 0
`
`50
`
`0
`
`B 350
`
`300
`
`Cl 250
`E
`1: 200 '--
`.Ql
`s 150
`Ql
`...
`0
`E 100
`::::i
`I-
`
`50
`
`0
`
`~· control
`/-~
`
`/_./-
`
`/
`-~-------------
`
`/
`
`~---
`
`ICI 5 mg/w
`
`7
`
`14
`
`21
`
`28
`
`35
`
`Treatment Days
`
`, - I tumor wAiQht
`
`1 350
`'
`
`300
`
`- """' """ 1 "'
`
`200
`
`-< 150
`
`100
`
`50
`
`0
`
`Control
`
`ICI
`
`~--·~..........___
`------
`35
`
`CGS 10 µg/d
`
`14
`
`21
`
`28
`
`Treatment Days
`
`400
`Qtu'llorwe9ht 350
`
`~uter1.1sweig~1t
`
`300
`
`250
`
`150
`
`. 100
`
`50
`
`r ~
`
`Ol
`E
`:E
`.Ql
`
`Ul
`
`2
`,SJ
`::i
`
`Ol
`E
`1:
`.!:!l
`s
`Ql
`"'
`...
`::::i
`.l!l
`::::i
`
`Ol 300 ~
`E
`250 L
`
`.:i::
`Cl
`'Qi
`s
`0
`E
`::::i
`I-
`
`200
`
`150
`
`100
`
`50
`
`0
`
`Control
`
`CGS
`
`ICI
`0.7mg/w
`
`ICI
`0.07mg/w
`
`0
`
`Figure 1. The effect of antiestrogen, ICI 182.780 on tumor and
`uterine wet weight in the nude mouse model. Groups of 4 mice
`with tumors of MCF-7 breast cancer cells transfected with the
`aromatase gene were injected sc with the anticstrogen, TCI
`182,780, 5 mg/mouse/week. or vehicle. A. Tumors were mea(cid:173)
`sured weekly and the percentage change in volume calculated.
`B. After 35 days of treatment. mice were sacrificed. tumors and
`uteri were weighed. Values, mean± SE. are significantly differ(cid:173)
`ent from control ** p < 0.01.
`
`Figure 2. The effect of antiestrogcn ICI 182,780 and aromatase
`inhibitor, letrozole on tumor and uterme wet weight 111 the nude
`mouse model. Groups of 4 mice were injected sc with !CI 0.7 mg
`or 0.07 mg in oil once per week, with letrozolc (CGS) 10 ~Lg in
`0.3% HPClmouse/day, or with 0.3% HPC vehicle. A. Tumors
`were measured \Veekly and the percentage change in volume cal(cid:173)
`culated. B. After 35 days of treatment, mice were sacrificed. and
`tumors and uteri were weighed. Values, mean ± SE, are signif(cid:173)
`icantly different from control ''' p < 0.05: ''* p < 0.01.
`
`These cells synthesize endogenous estrogen which
`is sufficient to stimulate their proliferation into tu(cid:173)
`mors and maintain the uterus in these animals. In
`the present study, we have used this model to in(cid:173)
`vestigate the effects of the aromatase inhibitors le(cid:173)
`trozole and anastrozole, and the steroidal antiestro(cid:173)
`gcns faslodex (ICI 182,780) and tamoxifen, on es(cid:173)
`trogen target tissues, mammary tumors and the
`uterus.
`
`Materials and methods
`
`Athymic mice
`
`Female BALB/c athymicmice 4-6 weeks of age (20--
`22 g bodyweight) were obtained from NCI. Freder(cid:173)
`ick, MD. The animals were huuseu i11 a pathugen(cid:173)
`free environment under control led conditions of
`light and humid1ty and received food and water nd
`libitwn. Ovariectomy was carried out under fluoro(cid:173)
`thane anesthesia 1-3 days before cell inoculation.
`
`MYLAN PHARMS. INC. EXHIBIT 1014 PAGE 3
`
`

`
`66
`
`Qing Lu et al.
`
`600 I
`600
`550 .
`500
`450
`400
`350
`300
`/
`JI(
`250
`200 /~/
`
`- 150
`
`/
`
`100
`50 -
`0
`
`A
`Cl)
`E
`:i
`0
`>
`'-
`0
`E
`:::l
`t-
`~
`t-
`0
`Q)
`Ol
`c
`ro
`.!::.
`u
`*'
`
`.... .-. control
`~-~-_,/',/
`·-·-·--"-·--· ·-·-··-
`
`,
`
`L
`
`TAM 60 µg/d
`CGS 10 µg/d
`CGS 60 µg/d
`
`A
`Q)
`E
`:J
`0
`>
`5
`E
`:J
`I-
`
`~
`0
`Q)
`Ol
`c:
`"'
`.c
`(.)
`?fl.
`
`160
`
`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`0
`
`7
`
`14
`
`21
`
`28
`
`Treatment Days
`
`control
`
`Arimidex 10 µg/d
`
`Arimidex 60 µg/d
`
`ICI 5 mg/w
`
`250 -
`
`B 350 .
`~ 300 -
`Ol
`$
`:E:
`Ol 200 -
`~
`150 -
`'-
`0
`E 100 '_
`:::l
`t-
`
`50 ~-
`
`0
`
`uterus weight
`
`T
`
`_l
`
`c= tumor weight J 350
`300 -Ol
`_§,
`:c
`200 Ol
`'iji
`150 s
`100 2
`2
`:::::>
`
`250
`
`50
`
`<J)
`
`control Arimidex Arimidex
`1 Oµg/d
`60µg/d
`
`ICI
`5 mg/w
`
`0
`
`0
`
`7
`
`14
`
`21 2fl
`
`35
`
`42
`
`49
`
`56
`
`Treatment Days
`
`1120
`
`tumor weight
`
`~ uterus weight
`
`100
`
`80 -
`en
`E
`:c
`60 ~
`2
`40
`2
`I
`::::i
`20
`
`Ol
`
`rl)
`
`Control
`
`ccs
`10µg/d
`
`cos
`60µg/d
`
`TAM
`60µg/d
`
`0
`
`B
`
`OJ
`E
`.E
`Ol
`·a;
`s:
`5
`E
`::J
`I-
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`Fi15ure 3. The effect uf antiesllogen tamoxifen and aromatase in(cid:173)
`hibitor letrozole on tumor and uterine wet weight in the nude
`mouse model. Groups of 4 mice were injected sc daily with letro(cid:173)
`zole (CGS) 10 µg/mouse/uay or 60 µgimuu,e/uay. larnuxifen 60
`µg/mouse/day. or vehicle. A. Tumors were measured weekly and
`the percentage change in volume calculated. B. After 56 days of
`treatment. mice were sacrificed, and rumors and uteri were
`weighed. Values. mean SE, are significantly different from
`control* p< 0.05. ** p< 0.01; CGS lOµgvs. TAM- p < 0.05; CGS
`60 µg vs. TAM ' p < 0.01.
`
`Table 1. The effect of letrozole and tamoxifen on tumor weight
`
`Figure 4. The effect of antiestrogen ICI 182,780 and aromatase
`inhibitor arimidex on tumor and uterine wet weight in the nude
`mouse model. Groups of 4 mice were injected sc daily with arimi(cid:173)
`dex !Oµg/mouse/day or 60 µg/mouse/day or ICI 182,780 (ICI)
`5mg/week sc or vehicle. A. Tumors were measured weekly and
`the percentage change in volume calculated. B. After 28 days of
`treatment. mice were sacrificed, and tumors and uteri were
`weighed. Values. mean SE, are signifieantly different from
`control* p < 0.05.
`
`Treatment
`
`vehicle
`vehicle
`tamoxifen
`lctrozolc
`letrozole'
`
`Treatment
`Days
`
`l
`56
`56
`56
`35-56
`
`Mice
`n
`
`4
`2
`,,
`4
`2
`
`Tumors
`n
`
`21
`17
`26
`22
`14
`
`Tumor
`mg wet weight'
`
`b 53.54 + 7 ~1
`226.3 + 31.10
`b 55.88 ± 12.20
`20.58 ± 2.09
`b 74.64 9.10
`
`Groups of four mice were injecteu sc with tamoxifen or letrozole (60 µg/mouse/uay) or vehicle. One gruuµ uf vdiicle-ueateJ mice were
`autopsied on Day 1. and tumors were removed and weighed. All other mice were autopsied on Day 56 of treatment.' Mean SE: b values
`are significantly different from control (p < 0.01). 'Two mice in the control group were crossed over to letrozole treatment on Day 21.
`
`MYLAN PHARMS. INC. EXHIBIT 1014 PAGE 4
`
`

`
`A
`
`350
`
`300
`
`250
`
`Control
`
`/ / /
`
`/
`
`/'"~
`
`TAM
`
`<ll
`E
`:::>
`0
`JI/,/ y-
`>
`i5
`/,./
`E
`:;i
`I-
`200.
`/ / / /
`·;!/
`19
`~
`I
`~/· • Arimidex
`0 1'0~/0
`<ll
`/~--*/---~
`Ol
`r::
`'1l
`100 ~~-
`.i= u
`
`Aromatase inhibitors and antiestrogens in nude mouse model
`_____ ,.
`
`67
`
`ulated sc in four sites with 0.1 ml of the cell suspen(cid:173)
`sion. Animals were then injected sc daily with 0.1
`mg androstenedione/mouse. Growth rates were de(cid:173)
`termined by measuring the tumors with calipers ev(cid:173)
`ery week. Tumor volumes were calculated accord(cid:173)
`
`ing to the formula 4/3xnxr12xr2 (r1<r2).
`
`Treatment
`
`Treatment began 21 to 35 days after androstene(cid:173)
`dione injections when tumors had reached a mea(cid:173)
`surable size. Mice were treated daily with sc injec(cid:173)
`tions of aromatasc inhibitors, lctrozolc ( CGS
`20,267) (kindly provided by Dr. Ajay Bhatnagar,
`Novartis. Basel Switzerland) and arimidex (kindly
`provided by Dr. Michael Dukes, Zeneca Pharma(cid:173)
`ceuticals, Macclesfield, UK) or the antiestrogen ta(cid:173)
`moxifen in 0.3 % hydroxypropylcellulose (HPC).
`The antiestrogen ICI 182,780 was injected in oil
`once per week (kindly provided by Dr. A Wakeling,
`Zeneca Pharmaceuticals, Macclesfield, UK). Con(cid:173)
`trol animals were given injections of vehicle (0.3%
`HPC, O.l ml/mouse/day) sc daily. The treatment
`lasted 4-5 weeks. Animals were autopsied 4 hours
`after the last injection. The uteri were removed.
`cleaned, and weighed. Multiple small tumors were
`removed from each inoculation site and their total
`weight determined. All compounds were very well
`tolerated and no adverse effects were observed.
`
`Statistics
`
`One way ANOVA was used to analyze the data.
`
`Results
`
`l. !Cl182, 780
`
`Treatment of a group of four mice with either ICI
`182.780 (5 mg/mouse/week; i.e., 35.7 mg/kg/week,
`sc) or vehicle was started 28 days after inoculation.
`There was no significant change in tumor volume in
`the ICI treated mice (789.99 ± 65.24 mm' to
`) during the 35 days of treat
`889.07 ± 155.45 rnm3
`ment. However. tumor volume in the control
`from 635.82 ± 57.15 mm3
`mice increased
`to
`
`~~
`
`50 i
`0
`
`B
`
`900
`
`800
`Ol 700
`E
`600
`
`i
`s:
`i5
`E
`:;i
`I-
`
`!JOO
`
`400
`
`300
`200
`
`100
`
`0
`
`7
`
`._[_______
`14
`
`21
`
`28
`
`35
`
`Treatment Days
`
`=:::J tumor weight
`~ uterus weight
`
`900
`800
`
`700
`
`600
`
`500
`
`400
`
`Ol
`E
`.E
`"' 'iii
`s:
`"'
`300 ~
`::J
`
`200
`
`Control
`
`Arimidex
`
`TAM
`
`100
`
`0
`
`Figure 5. The effect of antiestrogen tamoxifen and aromatase in(cid:173)
`hibitor arimidex on tumor and uterine weight in the nude mouse
`model. Groups of 5 mice were injected sc daily with tamoxifen
`3µg/mouse/day 01 a1 imidex 5µg/mouse/day u1 vdride. A. Tu(cid:173)
`mors were measured weekly and the percentage
`in vol(cid:173)
`ume calculated. B. After 35 days of treatment, mice were sacri(cid:173)
`ficed. LUmors and uleri were weighed. Values, mean ± SE. are
`significantly different from control p < 0.05;
`p < 0.01; arimi(cid:173)
`dex vs. TAM - p < 0.05:-+ p < 0.01.
`
`Cell culture and inoculation to athymic mice
`
`As reported previously [24], we used MCF-7 cells
`stably transfected with the human placental aroma(cid:173)
`tase gene (MCF-7 cA). The cells were cultured in Ea(cid:173)
`gle's minimum essential medium containing 5% fe(cid:173)
`tal bovine serum and neomycin (600 µg/ml; GIB(cid:173)
`CO, Bethesda, MD). The culture medium was
`changed twice weekly. Subconfluent MCF-7cA cells
`were scraped into Hank's solution and centrifuged
`at 1,000 rpm for 2 min at 4° C. The cells were then
`resuspended in Matrigel (10 mg/ml) (kindly provid(cid:173)
`ed by Dr. Hynda Kleinman. NCI) to make a cell sus(cid:173)
`pension of 2-5 107 cells/ml. Each mouse was inoc-
`
`MYLAN PHARMS. INC. EXHIBIT 1014 PAGE 5
`
`

`
`68
`
`Qing Lu et al.
`
`2053.19 ± 605.98 mm 3 (222.11 % ). The mean tumor
`weight of the ICI group (112.91
`23.15 mg) was sig(cid:173)
`nificantly
`less
`than of
`the
`control mice
`(276.69 ± 53.05 mg) (p < 0.01) (Fig. 1). The mean
`uterine weight of the ICI group (8.83 ± 1.02 mg) was
`also signifieantly
`less
`than of
`the controls
`(59.36 ± 7.80 mg) (p < 0.01) at the end of the treat(cid:173)
`ment period. Thus, ICI 182,780 was effective in re(cid:173)
`ducing growth of MCF-7 CA tumors. This compound
`also blocked the effect of endogenous estrogen on
`the uterine weight in OVX mice supplemented with
`androstenedione.
`
`2. Letrozale vs ICl 182, 780
`
`Thirty-five days after inoculation. a group of four
`mice received 10 µg/mouse/d letrozole sc, 0.7 mg/
`mouse/week ICI 182.780 sc, 0.07mg/mouse/week
`ICI (this dose is equivalent to the letrozole dose or
`vehicle. After 35 days of treatment. tumor volumes
`in the letrozole 10 µg/d treated animals showed a
`decrease in volume (from 523.45 ± 45.87 mm3 to
`261.73 ± 34.12 mm3
`). Tumor volumes in the ICI 0.07
`mg/wand ICI 0.7 mg/w treated mice changed from
`756.34 87.56 mm3 to 1361.41 134.75 mm3 and
`from 514.86 ± 76.12 mm3 to 771.23 ± 103.45 mm3
`, re(cid:173)
`spectively. Tumor volume in the control mice in(cid:173)
`746.34 ± 121.34 mm'
`to
`creased
`from
`1865.57 ± 156.89 mm3 (267.96% ). The mean tumor
`weights for the letrozole (20.37 ± 2.19 mg) and ICI
`0.7 mg (100.71±18.55 mg) treated groups were sig(cid:173)
`nificant less than for the controls (285.75 ± 74.43
`mg) (p < U.Ul) (Fig. 2). The mean uterine weights
`for the letrozole (15.20 ± 1.51 mg) and ICI 0.7mgh.v
`(19.3 ± 3.04 mg) groups were similar and signifi(cid:173)
`cantly less than for the controls (53.87 ± 3.19 mg)
`(p < 0.01) (Fig. 2). Both letrozole and ICI 182,780
`were effective in reducing the growth of MCF-7Ca
`tumors, although a greater reduction in tumor
`growth occurred in the letrozole group.
`
`3. Letrozole vs Tamoxzfen
`
`Thirty days after inoculation, groups of four mice
`received letrozole (10 µg/mouse/d, sc, 60 µg/
`mouse/d, sc, this dose was used previously [25]), or
`60 µg/mouse/d, sc tamoxifen. Tumor volumes in the
`
`letrozole 10 µg and 60 µg treatment groups showed
`a marked decrease in volume after 3 weeks of treat(cid:173)
`ment (from 385.84 ± 89.84 mm3 to 308.64 73.68
`425.68 ± 73.92 mm3
`mm3
`and
`from
`to
`•
`298.45 ± 69.36 mm3
`, respectively). Tumor volumes
`the
`tamoxifen
`group
`changed
`from
`in
`401.12 ± 128.96 mm3 to 501.44 ± 97 .52 mm3
`, whereas
`volumes in the control animals increased from
`419.04 101.52 mm3
`to 2430.40 ± 422.56 mm3
`( 479.99%) during treatment (Fig. 3). After 56 days
`of treatment, the mice were sacrificed, and the tu(cid:173)
`mors and the uterus were removed and weighed.
`The mean tumor weight of the control group was
`greater than those of tumors from both the letro(cid:173)
`zole 10 µg and 60 µg treatment groups: 87.66 ± 8.00
`mg versus 28.30 ± 8.30 mg, and 20.83 ± 2.08 mg, re(cid:173)
`spectively (p < 0.01) (Fig. 3). The mean tumor
`weight of the control group was also greater than
`that of tumors from the tamoxifen treatment group
`(55.88 ± 12.20 mg; p < 0.05) (Fig. 3). In addition, the
`mean tumor weights for both letrozole 10 µg and 60
`µg were less than for the tamoxifen group (p < 0.05
`and p < 0.01), respectively (Fig. 3). The mean uLer(cid:173)
`ine weights of the control group (48.66 7.60 mg)
`and of the tamoxifen group (54.33 ± 2.84 mg) were
`greater than those of the letrozole 10 µg and 60 µg
`groups (18.98 ± 2.22 mg and 19.19 ± 2.62 mg;
`p < 0.01 ). Thus, both letrozole and tamoxifen inhib(cid:173)
`ited the growth of the tumors, but letrozole was
`more effective than tamoxifen. However, in these
`experiments tamoxifen was either uterotrophic or
`did not block the effects of estrogen produced by
`the tumor in the ovariectomized nude mice.
`In a further experiment, groups of five mice were
`injected for 56 days with either letrozole, tamoxi(cid:173)
`fen, or vehicle. Tumors were removed and weighed
`from a group of mice sacrificed on the first day of
`treatment. After 21 days, two mice from the control
`group were crossed over to letrozole treatment for a
`further 35 days. As shown in Table 1, tumor weights
`were significantly less than controls in all treatment
`groups (p < 0.05). An additional finding was that
`the mean tumor weight of the 60µg letrozole group
`was less than that of the group sacrificed on Day l,
`although the difference was not statistically signif
`icant. Also. 35 days of treatment was sufficient to
`
`MYLAN PHARMS. INC. EXHIBIT 1014 PAGE 6
`
`

`
`Aromatase inhibitors and antiestrogens in nude mouse model
`
`69
`
`inhibit tumor growth even in relatively large tu(cid:173)
`mors.
`
`icant difference in body weights of the treated ani(cid:173)
`mals and the control group in any of the experi(cid:173)
`ments.
`
`4. Arimidex vs. !CI
`
`Twenty-one days after inoculation, groups of four
`mice received arimidex (10 µg/mouse/d, sc and 60
`µg/mouse/d, sc), ICI 182,780 (Smg/mouse/w, sc) or
`vehicles (control). The treatment continued for 28
`days. The tumor volume for the control group in(cid:173)
`creased from 801.80±82.71mm3 to1222.46±177.27
`mm3 (185.50%) (Fig. 4). The mean tumor volume
`for the 10 µg/day and 60 µg/day arimidex groups
`changed from 664.80 187.33to155.65 ± 101.69 and
`from 598.58 ± 119.69 mm3 to 376.25 ± 94.27 mm3
`, re(cid:173)
`spectively (Fig. 4 ). The tumor volume for the arimi(cid:173)
`dex groups remained the same. The mean tumor
`weights for arimidex 10 µg and ICI treated animals
`were significant less than for the control group
`(p < 0.05) (Fig. 4 ). Thus, at the doses used, arimidex
`and ICI 182,780 inhibited the growth of these tu(cid:173)
`mors to about the same extent.
`
`5. Arimidex vs. tamoxifen
`
`Twenty eight days after inoculation, groups of five
`mice received low doses of arimidex (5 µg/mouse/d,
`sc), and tamoxifen (3 µg/mouse/d, sc). The controls
`received vehicle. Treatment continued for 35 days.
`Tumor volume in the arimidex treated group
`7776.20 80.03 mm3
`changed
`from
`to
`1247.97 ± 83.52 mm3 (Fig SA). Tumor volume of the
`control group increased from /34.62 ± 22:i.'/4 mmJ
`to 2405.00 ± 475.29 mm3 (327.38%) (Fig.SA). The
`mean tumor weight for
`the arimidex group
`( 452.88 78.88 mg) was significantly less than for
`the control group (p < 0.05) (Fig.SB), whereas the
`mean
`tumor weight
`for
`tamoxifen was
`661.88 ± 58.31 mg which was not significantly differ(cid:173)
`ent from the control group (Fig. SB). The uterine
`weight for the arimidex group (28.00 7.16 mg) was
`significantly less than for the tamoxifen and control
`groups (68.2.5 ± 13.98 mg) (p < 0.01) (Fig. SB). Thus,
`arimidex was more effective in reducing tumor and
`uterine weight than tamoxifen.
`All compounds were well tolerated and no ad(cid:173)
`verse effects were observed. There was no signif-
`
`Discussion
`
`Ovariectomized nude mice inoculated with MCF7
`human breast cancer cells transfecte.d with aroma(cid:173)
`tase (MCF-7 cA) synthesize estrogens, which en(cid:173)
`hance tumor growth and maintain the uterine
`weight comparable to that of the intact mice. This
`model offers an opportunity to study the effects of
`antiestrogens and aromatase inhibitors in the same
`system. We have previously shown that these tu(cid:173)
`mors respond to tamoxifen and aromatase inhib(cid:173)
`itors [24]. In the current studies, we have used com(cid:173)
`parable doses of the compounds in each experiment
`to determine their efficacy in vivo. In addition, the
`doses selected produced only partial responses
`rather than complete tumor suppression, in order to
`achieve a more accurate comparison. The antiestro(cid:173)
`gens, tamoxifen and ICI 182,780, were effective in
`reducing tumor growth in these nude mice, al(cid:173)
`though ICI at 5 mg injected once per week did not
`appear to be any more effective than tamoxifen
`(60 µg/day). There was no significant difference be(cid:173)
`tween the uterine weights of the controls and ta(cid:173)
`rnoxifen treated animals, consistent with previous
`findings of specific effects reported for the agonist/
`antagonist actions of tamoxifen [9]. However, ICI
`blocked the effects of estrogen on the mouse uterus,
`suggesting that there may be a difference in the sen(cid:173)
`sitivity of the tumor and the uterus to these anti(cid:173)
`estrogens.
`At similar concentrations, the antiestrogens were
`not as effective as the two aromatase inhibitors, ari(cid:173)
`midex and letrozole, in preventing the growth of tu(cid:173)
`mors in this model. Because ICI is a potent anties(cid:173)
`trogen, greater efficacy in vivn was therefore ex(cid:173)
`pected. One explanation for our results might be
`that the compound has less favorable pharmacoki(cid:173)
`netics when injected once a week into the mouse,
`compared to daily injections of the other com(cid:173)
`pounds. All of these compounds are likely to induce
`greater tumor suppression at higher doses. Also,
`other mechanisms of the compounds may contrib-
`
`MYLAN PHARMS. INC. EXHIBIT 1014 PAGE 7
`
`

`
`70
`
`Qing Lu et al.
`
`ute to their antitumor effects at higher doses. How(cid:173)
`ever, it is interesting to note that letrozole not only
`suppressed tumor growth but appeared to cause tu(cid:173)
`mor regression in the nude mice. Thus, tumor
`weights after 56 day of letrozole treatment were
`60% lower than tumor weights of a group of mice
`sacrificed on the day treatment began (p < 0.05)
`(Table 1). A similar Ln::nd in n::ducliun of Lumur vol(cid:173)
`umes occurred with arimidex. It is presently unclear
`why the lower dose of arimidex was more effective
`than the higher dose in reducing tumor volume and
`weight. Letrozole also caused marked suppression
`of the growth of relatively large tumors in the ex(cid:173)
`periment where two mice were crossed over from
`the control group to letrozole treatment. After only
`3 weeks of treatment, the mean tumor weight was
`74.68 ± 9.1 mg in comparison to 226.3 ± 31.1 mg for
`tumors of the control mice.
`In conclusion, the data presented indicate that
`the aromatase inhibitors letrozole and arirnidex nre
`highly effective in suppressing tumor growth and
`are more effective at comparable doses than the an(cid:173)
`tiestrogens tamoxifen and ICI 182,780 in this mouse
`model. Estrogen production in this intratumoral
`aromatase model is sufficient to maintain the
`weight of the uterus in the ovariectomized mouse
`similar to that of the intact animal. Although ta(cid:173)
`moxifen did not reduce the weight of the uterus, the
`antiestrogen ICI 182,780 and the aromatase inhib(cid:173)
`itors prevented the effect of estrogen on the uterus,
`suggesting that these compounds may not cause en(cid:173)
`dometrial hyperplasia in patients as reported for ta(cid:173)
`moxifen [8]. These aromatase inhibitors may not
`only benefit patients who have relapsed from ta(cid:173)
`moxifcn, but may be more effective in patients as
`first line agents for suppressing the effects of estro(cid:173)
`gen.
`
`Acknowledgments
`
`This work was supported by NIH grant CA 62483.
`
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